The main objective of our current grant has been achieved by demonstrating the mechanism of metabolic activation of 7,12- dimethylbenz[a]anthracene (DMBA). The first goal in the proposed continuation is to demonstrate the validity of our hypothesis that one- electron oxidation is the major pathway of activation for the most potent polycyclic aromatic hydrocarbons (PAH). To study the mechanism of activation of representative carcinogenic methylbenz[a]anthracenes (MBA), we propose to identify and quantify the DNA adducts of 3- methylcholanthrene (MC), 6-MBA, 7-MBA, 8-MBA and 12-MBA formed in vitro with activation by horseradish peroxidase or rat liver microsomes and in vivo in mouse skin or rat mammary gland (Project 1). Our second goal is to elucidate the biological significance of PAH-DNA adducts. To begin, we will identify the sites of adduct formation and mutation in the supF gene in vitro with activation of benzo[a]pyrene (BP), DMBA and dibenzo[a,l]pyrene (DB[a,l]P) by one-electron oxidation or formation of diol epoxides, and identify the sites of mutation in the lacI gene in vivo in transgenic B6C3F1 mice after treatment with BP, DMBA or DB[a,l]P (Project 4). A third goal is further development of analytical and structural methods for DNA adducts at the subpicomole level, by using fluorescence line narrowing spectroscopy (FLNS) (Project 3) and fast atom bombardment tandem mass spectrometry (FAB MS/MS) (Project 2). Improvements in FAB MS/MS offer the potential for separating and identifying femtomolar levels of modified nucleosides, nucleotides and, possibly, small oligonucleotides. Improvements in laser-induced fluorescence instrumentation should increase the detectability for PAH- DNA adducts to less than or equal to 100 attamole and allow coupling to separation techniques. FLNS will also be used to study the dependence of DNA repair on adduct site conformation (intercalated, base-stacked or exterior). The role of PAH DNA physical complexes leading to formation of adducts by one electron oxidation will be addressed by using FLN (project 3). From our collective efforts we expect to develop a comprehensive picture of metabolic activation of unsubstituted and methyl-substituted PAH starting with the initial physical complex of the PAH with DNA, continuing to the formation of various DNA adducts, and ending with the role of both stable and depurination adducts in mutagenic events that may play a role in carcinogenesis. The quest for understanding PAH carcinogenesis is 60 years old. We think we have the know-how and necessary technology to gain a clear picture of the mechanism of metabolic activation of PAH and to begin to understand the biological significance of PAH-DNA adducts.